Structural Basis for the Unusual Qy Red-Shift and Enhanced

Article pubs.acs.org/biochemistry

Structural Basis for the Unusual Qy Red-Shift and Enhanced Thermostability of the LH1 Complex from Thermochromatium tepidum Long-Jiang Yu,†,§ Tomoaki Kawakami,† Yukihiro Kimura,‡ and Zheng-Yu Wang-Otomo*,† †

Faculty of Science, Ibaraki University, Mito 310-8512, Japan Department of Agrobioscience, Graduate School of Agriculture, Kobe University, Nada, Kobe 657-8501, Japan

‡

S Supporting Information *

ABSTRACT: While the majority of the core light-harvesting complexes (LH1) in purple photosynthetic bacteria exhibit a Qy absorption band in the range of 870−890 nm, LH1 from the thermophilic bacterium Thermochromatium tepidum displays the Qy band at 915 nm with an enhanced thermostability. These properties are regulated by Ca2+ ions. Substitution of the Ca2+ with other divalent metal ions results in a complex with the Qy band blue-shifted to 880−890 nm and a reduced thermostability. Following the recent publication of the structure of the Ca-bound LH1-reaction center (RC) complex [Niwa, S., et al. (2014) Nature 508, 228], we have determined the crystal structures of the Sr- and Ba-substituted LH1-RC complexes with the LH1 Qy band at 888 nm. Sixteen Sr2+ and Ba2+ ions are identified in the LH1 complexes. Both Sr2+ and Ba2+ are located at the same positions, and these are clearly different from, though close to, the Ca2+-binding sites. Conformational rearrangement induced by the substitution is limited to the metalbinding sites. Unlike the Ca-LH1-RC complex, only the α-polypeptides are involved in the Sr and Ba coordinations in LH1. The difference in the thermostability between these complexes can be attributed to the different patterns of the network formed by metal binding. The Sr- and Ba-LH1-RC complexes form a single-ring network by the LH1 α-polypeptides only, in contrast to the double-ring network composed of both α- and β-polypeptides in the Ca-LH1-RC complex. On the basis of the structural information, a combined effect of hydrogen bonding, structural integrity, and charge distribution is considered to influence the spectral properties of the core antenna complex.

L

respectively, a large number of studies have been conducted to elucidate the structures of LH1 and LH1-RC complexes.17−21 Despite intensive efforts, the resolutions of the structures of these complexes had for a long time remained low.22,23 Recently, the native Tch. tepidum LH1-RC complex has been crystallized,24 and its structure has been determined at 3.0 Å resolution.25 This core complex contains 36 polypeptides and 80 cofactors with a total molecular weight of 390 kDa. LH1 reveals a closed and slightly elliptical ring with 16 αβ subunits encircling the RC. The bacteriochlorophyll (BChl) a molecules in LH1 are aligned with the special pair in RC on the same plane that parallels the membrane surface. The shortest distance between the central Mg atom of the special pair BChl a and that of LH1 BChl a is ∼38 Å. This spatial arrangement is considered as the optimized configuration for facilitating efficient migration of the excitation energy from LH1 to RC. Thirty-two BChl a molecules in the LH1 form a partially overlapping ring with an average Mg−Mg spacing of

ike most organisms, photosynthetic bacteria have evolved through adaptation to their living environments by utilizing surrounding natural resources. A typical example comes from the thermophilic purple sulfur bacterium Thermochromatium tepidum1 isolated from a hot spring in Yellowstone National Park that is rich in mineral calcium carbonate.2 This bacterium can grow at an optimal temperature of 48−50 °C with an upper limit of 58 °C. Many enzymes and proteins purified from Tch. tepidum exhibit thermostability higher than those of their mesophilic counterparts.3−6 Among these, the core light-harvesting complex (LH1) has been demonstrated to acquire Ca2+ ions for its enhanced thermostability.7 Spectroscopically, Tch. tepidum LH1 exhibits a characteristic Qy absorption band at 915 nm.8,9 This contrasts with the majority of the LH1 complexes from other species that show the Qy band in the range of 875−890 nm and provides a model system for studying “uphill” energy transfer. The largely red-shifted Qy transition of Tch. tepidum LH1 is attributed to binding of Ca2+ to the C-terminal domains of the LH1 polypeptides.10,11 Following the high-resolution structural determinations of the bacterial reaction center (RC)12−14 and peripheral lightharvesting complex (LH2)15,16 achieved 30 and 20 years ago, © 2016 American Chemical Society

Received: July 21, 2016 Revised: August 24, 2016 Published: November 16, 2016 6495

DOI: 10.1021/acs.biochem.6b00742 Biochemistry 2016, 55, 6495−6504

Article

Biochemistry Table 1. Crystallographic Data Collection and Refinement Statistics of the Sr- and Ba-LH1-RC Complexes Sr-LH1-RC

Ba-LH1-RC

beamline wavelength (Å) resolution range (Å) space group cell dimensions a, b, c (Å) β (deg) Rsym I/σ(I) completeness (%) redundancy

BL44XU/SPring-8 0.76 (Sr peak) 50−3.5 (3.56−3.50) P21 164.2, 147.8, 210.3 107.8 0.10 (0.72) 17.3 (1.8) 99.1 (99.7) 7.3 (5.8)

resolution range (Å) no. of reflections Rwork/Rfree (%) no. of atoms average B factor (Å2) rmsd bond lengths (Å) bond angles (deg) Ramachandran plot (%) favored allowed outlier

50−3.5 216518 26.2/29.9 51820 166

Data Collectiona BL44XU/SPring-8 BL44XU/SPring-8 0.78 (Sr remote) 0.90 50−3.5 (3.56−3.50) 50−3.3 (3.36−3.30) P21 P21 164.4, 149.1, 210.0 164.9, 148.9, 210.2 108.4 108.2 0.10 (0.74) 0.13 (0.63) 19.0 (1.7) 20.4 (1.7) 98.7 (99.2) 97.3 (76.8) 7.1 (5.6) 6.4 (3.2) Refinement 50−3.5 50−3.3 219756 142691 26.1/29.1 27.1/30.9 51820 51881 182 158

0.034 2.58

0.037 2.65

87.7 10.2 2.1

87.8 10.2 2.0

a

Values in parentheses refer to those of the highest-resolution shell.

BL17A/PF 0.98 50−3.3 (3.38−3.30) P21 163.8, 148.6, 210.0 108.1 0.43 (6.49) 8.6 (0.77) 92.6 (90.1) 22.8 (23.2)

BL17A/PF 1.50 (Ba detection) 56−4.5 (4.62−4.50) P21 166.4, 150.3, 211.0 108.1 0.22 (1.88) 11.0 (1.7) 99.9 (99.9) 18.8 (18.9)

50−3.3 133049 27.9/31.3 51881 161

50−4.5 114512 34.6/35.8 51820 207

0.035 2.26

0.032 2.16

0.033 2.64

87.6 10.1 2.3

88.5 8.9 2.6

88.5 9.7 1.8

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MATERIALS AND METHODS Preparation of the Sr- and Ba-LH1-RC Crystals. The procedures for preparing Sr- and Ba-LH1-RC complexes were similar to that used for the native LH1-RC complex from Tch. tepidum strain MC24,25 with minor modifications. The Sr2+ and Ba2+ ions were introduced in the step of DEAE anion-exchange chromatography. The LH1-RC complex-rich component after treatment with lauryldimethylamine N-oxide was dissolved in 20 mM Tris-HCl (pH 8.5) containing 1.0% n-dodecyl β-Dmaltopyranoside (DDM) and loaded on a DEAE column (Toyopearl 650S, TOSOH) equilibrated with 20 mM Tris-HCl (pH 7.5) and 0.05% DDM at 4 °C. The LH1-RC fractions were eluted with a linear gradient of SrCl2 or BaCl2 from 10 to 25 mM. The partially substituted LH1-RC complexes were first concentrated with an Amicon Ultra filter (100K molecular weight cutoff, Millipore) and then diluted with 20 mM TrisHCl (pH 7.5) containing 0.8% n-decylphosphocholine (DPC) and 50 mM SrCl2 or BaCl2. To reduce the effect of DDM on crystallization, this procedure was repeated three times to make the DDM concentration less than 0.0005%. Crystallization of the Sr- and Ba-LH1-RC complexes and postcrystallization treatment were conducted following the same method that was used for the native LH1-RC complex,25 except using SrCl2 or BaCl2 instead of CaCl2. Suitably sized crystals were obtained at 20 °C (∼0.4 mm × 0.7 mm × 0.3 mm) after 2 weeks. Characterization of the Sr- and Ba-LH1-RC Crystals. Sr- and Ba-LH1-RC crystals were collected and dissolved in a 20 mM Tris-HCl (pH 7.5) solution containing 0.8% DPC and 50 mM metal ions. Absorption spectra were recorded on a Beckman DU-640 spectrophotometer. The content of the residual Ca2+ ions in the Sr- and Ba-substituted LH1-RC complexes was analyzed using a polarized Zeeman atomic absorption spectrophotometer (model Z-2010, Hitachi). Prior to the analysis, 5 mL of the redissolved LH1-RC complexes

8.77 Å. This distance is shorter than that of LH2, indicating a stronger coupling between the bacteriochlorins. Sixteen Ca2+ ions are identified in the LH1 complex from the crystal structure. They are distributed between the LH1 inner and outer rings and bind the α- and β-polypeptides to form a network that strengthens the whole LH1 structure. The structural feature explains well the increased thermostability of this complex.7 At the current resolution, the electron density map could be best interpreted by a structural model in which the Ca2+ is coordinated by α-Trp46, α-Asp49, α-Asn50, and the C-terminal β-Leu46 in the adjacent subunit.25 Structural evidence of a possible ubiquinone pathway through the closed LH1 ring is provided for the first time. The Ca2+ ions in Tch. tepidum LH1 can be replaced with other divalent metal ions (e.g., Sr2+ and Ba2+) during or after the purification process, resulting in a substituted LH1-RC complex with the LH1 Qy band in the range of 880−890 nm.10 The substitution is accompanied by a reduction in the thermostability of the LH1-RC complex to the same level as those of LH1-RC complexes purified from other mesophilic bacteria.7 This process is fully reversible as both the red-shifted LH1 Qy band and the thermostability can be fully restored by adding Ca2+ back to the complex. Among the various divalent metal ions tested, only Sr2+ can biosynthetically substitute for Ca2+ and support photosynthetic growth of the Tch. tepidum cells.26 Inspired by these unique properties and to elucidate the structural difference between the LH1-RC complexes with different LH1 Qy transitions, we have been trying to crystallize the metal-substituted LH1-RC complexes. Recently, crystals of Sr- and Ba-LH1-RC complexes have been obtained with good diffraction quality, and the structures of these complexes have been determined. In this study, the structural features are described and discussed in relation to the characteristic spectroscopy and thermodynamics. 6496


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Biochemistry (A513 = 4.0) was mixed with the same volume of 65% nitric acid (Suprapur, Merck), and the LH1-RC complexes were decomposed by heating the mixtures at 95 °C overnight followed by dilution with the ultrapure water treated with Chelex 100 resin (Bio-Rad). All the metal-free polypropylene vessels (DigiTube, SCP Science) were rinsed with 0.1 M HCl and/or the ultrapure water before being used. Diffraction Data Collection and Structural Analysis. Xray diffraction data were collected at BL41XU and BL44XU of SPring-8 (Hyogo, Japan), and BL1A, BL17A, and NE3A of the Photon Factory (Ibaraki, Japan) at 100 K. To identify the Sr2+and Ba2+-binding sites, data sets were acquired at three wavelengths for the Sr-LH1-RC complex and at two wavelengths for the Ba-LH1-RC complex (Table 1). The diffraction data were processed, integrated, and scaled using the HKL200027 and XDS packages.28 Structure determination was performed by molecular replacement method using the Phaser program29 in PHENIX.30 The search model was the crystal structure of the native LH1-RC complex [Protein Data Bank (PDB) entry 3WMM] at 3.0 Å from Tch. tepidum25 with the Ca2+ ions, lipid molecules, and solvent molecules removed. Positions of the Sr and Ba atoms were determined from anomalous difference Fourier maps.31,32 The Sr- and Baincorporated models were refined and modified using the Phenix.ref ine31 and Coot programs,33 respectively. Positional and isotropic displacement parameters were refined in the resolution range of 45−3.3 Å. The final models were refined to 3.3 Å resolution with Rfree values of 30.9 and 31.3% for the Srand Ba-LH1-RC complexes, respectively (Table 1). Coordinates of the crystal structures have been deposited in PDB as entries 5B5M (Sr-LH1-RC) and 5B5N (Ba-LH1-RC).

Figure 1. Absorption spectra of the metal-substituted LH1-RC complexes (dissolved crystals): red, Sr-LH1-RC; blue, Ba-LH1-RC. For comparison, absorption spectra of the Ca-LH1-RC complex (dissolved crystals) from Tch. tepidum and the LH1-RC complex from Alc. vinosum (purified solution) are shown by dashed and solid black curves, respectively. The inset displays an expanded view of the Qy region. All spectra were normalized to give the LH1 Qy intensity of 1.0.

To examine the extent of substitution, we have measured the Ca contents in the Sr- and Ba-LH1-RC complexes by atomic absorption spectroscopy (Table S1). The native Tch. tepidum LH1-RC complex was used as a control, in which 18 ± 1 Ca atoms per LH1-RC crystal were detected. This is consistent with the number of 17 Ca2+ ions as identified from the LH1-RC crystal structure.25 Approximately two Ca atoms per LH1-RC complex were present in the Sr2+- and Ba2+-substituted solution samples prior to crystallization, and no Ca was detected in the final redissolved crystals within experimental error. The result suggests that the substitution continued during the crystallization process and the crystals used in diffraction data collection were fully substituted. Identification of the Sr2+- and Ba2+-Binding Sites. Anomalous difference Fourier maps obtained at wavelengths of 0.76 and 1.5 Å revealed strong electron densities for the Sr- and Ba-LH1-RC crystals, respectively (panels a and c, respectively, Figure 2). The strong densities disappeared at 0.78 Å (remote) for the Sr-LH1-RC complex (Figure 2b) and were largely reduced at 0.98 Å for the Ba-LH1-RC complex (Figure 2d), confirming that they are truly from the Sr2+ and Ba2+ ions, respectively. Both Sr2+ and Ba2+ are located at essentially the same positions in the substituted LH1-RC complexes. Sixteen Sr2+ (Ba2+) sites are identified in the C-terminal regions of LH1 at clearly different positions from the Ca2+-binding sites. Two Sr2+ (Ba2+) ions exist at the interface between the RC and LH1, and one Sr2+ (Ba2+) is buried in the RC but at a position different from the Ca2+ site in the Ca-LH1-RC complex. Because the positions of Sr2+ and Ba2+ are essentially the same in the substituted LH1-RC complexes, we use the structure of the Sr-LH1-RC complex for description. Sixteen Sr2+ ions are uniformly distributed on the periplasmic side of the LH1 inner ring composed of α-polypeptides (Figure 3a). The Sr2+-binding sites in LH1 are close to, but clearly different from, the Ca2+-binding sites in the Ca-LH1-RC complex with an average Sr-Ca displacement of 5.4 Å. Because the Sr2+ sites are distant from the LH1 β-polypeptides, only the αpolypeptides are involved in metal coordination. Each binding

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RESULTS Preparation and Characterization of the Sr- and BaLH1-RC Crystals. Substitution of the Ca2+ in the native Tch. tepidum LH1-RC complex with Sr2+ or Ba2+ is a slow process and can be monitored by the blue-shift of the LH1 Qy transition.10 In some cases, it took several days for completion, especially for the highly concentrated LH1-RC complexes. Partially substituted LH1-RC complexes were eluted at 25 mM Sr2+ and Ba2+ from DEAE chromatography with the LH1 Qy maxima shifted to 909 and 894 nm, respectively. Further blueshifts were promoted by incubating the LH1-RC complex in the Sr2+ or Ba2+ buffer at a higher concentration (50 mM) for several hours at 4 °C. The same buffers were used for crystallization. Figure 1 shows the absorption spectra of the redissolved Sr- and Ba-LH1-RC crystals. The LH1 Qy bands were observed at 888 nm for both Sr- and Ba-LH1-RC complexes. A notable feature is that the substitution with Sr2+ is much slower than that with Ba2+, although the final LH1 Qy transitions become the same (Figure S1). The Sr-substituted LH1-RC crystal shows a Qy peak at the same position as that obtained from the biosynthetically Sr2+-substituted LH1-RC crystal,26 indicating that the substitution has been completed. The absorption spectra of both Sr- and Ba-LH1-RC complexes are similar to that of the native LH1-RC complex of Allochromatium vinosum (Figure 1), a mesophilic counterpart and close relative of Tch. tepidum. It was noted that there was increased absorbance at 760 nm in the spectra of the redissolved Sr- and Ba-LH1-RC crystals. This was likely to be due to a small amount of free BChl a resulting from the crystal resolubilization process. 6497


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Overall Structures of the Sr- and Ba-LH1-RC Complexes. Both Sr- and Ba-substituted LH1-RC complexes form crystals belonging to space group P21 in which two complexes are contained in the asymmetric unit (61% solvent content). The overall structures are homologous to that of Ca-LH1-RC complex.25 Superimpositions of the protein structures of Srand Ba-LH1-RC complexes with that of the Ca-LH1-RC complex yielded root-mean-square deviations (rmsd’s) of 0.926 and 0.943 Å for all Cα atoms, respectively, suggesting that metal ion substitution did not significantly alter the overall conformation of the core complex. This is in agreement with the findings from circular dichroism and ATR-FTIR that there is no essential change in the secondary structures between the native and metal-substituted complexes.10,34 The refined structures of the Sr- and Ba-LH1-RC complexes are effectively identical to each other at the tertiary structure level with a rmsd of 0.193 Å for all protein atoms, consistent with the ATR-FTIR result showing that the structural changes induced by Sr and Ba substitutions are essentially the same.35 Sixteen pairs of the LH1 αβ-polypeptides encircle the RC forming a closed and slightly elliptical cylinder with the bound cofactors being 32 BChls a, 16 spirilloxanthins, and 16 Sr2+ or Ba2+ ions. All of the metal ions in the core complexes are located on the periplasmic side and aligned approximately on the same plane of the presumed membrane surface (Figure 5a). The metal ions in LH1 serve as linkers connecting the α-polypeptides only and forming an inner ring network without the involvement of βpolypeptides (Figure 5b). In contrast, both the α- and βpolypeptides in the Ca-LH1-RC complex are involved in the formation of the LH1 network where the inner (α) and outer (β) rings are connected by the Ca2+ ions (Figure 5c). Therefore, the structural feature of the Sr- and Ba-LH1-RC complexes can well account for their reduced thermostabilities relative to that of the Ca-LH1-RC complex and the higher stabilities relative to that of the metal-removed LH1-RC complex.7 The decrease in the degree of structural integrity is also considered one of the reasons for the Qy blue-shifts of the substituted LH1-RC complexes.36 Structural Differences between the Sr(Ba)- and CaLH1-RC Complexes. Although the overall structures of the Sr(Ba)- and Ca-LH1-RC complexes are highly similar, relatively large differences have been observed around the metal-binding sites in LH1. Figure 6 shows an overlay of two αβ-subunits in the Sr- and Ca-LH1-RC complexes by superimposition of the Cα atoms. Upon metal substitution, the loop region in the Cterminal domain of α-polypeptides (α-Thr42−α-Val53) undergoes structural rearrangement and the C-terminal short helix (α-Ser54−α-Lys61) becomes less well-defined. The C-terminal end of Sr(Ba)-LH1 β-polypeptides (β-Pro44−β-Leu46) turns outward with respect to that of the Ca-LH1 complex, indicating that the loss of metal binding leaves this portion more mobile. The local conformational changes upon Sr(Ba) substitution were also detected by ATR-FTIR measurements.35 Notable changes are also found for the BChls a in LH1, which may contribute to the observed Qy transition. Table 2 shows the comparisons of hydrogen bonds, relative distances, and dihedral angles of BChls a among the Sr-, Ba-, and Ca-LH1 complexes. The hydrogen bonds formed between the C3-acetyl group (O32) of BChl a and the tryptophan side chain (Nε1) of both α- and β-polypeptides tend to be longer in the Sr- and BaLH1 complexes, suggesting that the interactions become weaker upon metal ion substitution. In particular, substantial increases (∼0.55 Å) in H-bond lengths are observed between

Figure 2. Anomalous difference Fourier maps from the data sets collected at wavelengths of (a) 0.76 and (b) 0.78 Å for the Sr-LH1-RC complex and at (c) 1.5 and (d) 0.98 Å for the Ba-LH1-RC complex. Red meshes represent the contour levels at 3.5σ in panel a and 3.0σ in panel b. Magenta meshes represent a contour level of 3.8σ in both panels c and d. All structures are shown as top views from the periplasmic side of the membrane. The following color scheme is used for the proteins: LH1-α, green; LH1-β, blue; RC-C, cyan; RC-L, sky blue; RC-M, yellow-orange; RC-H, orange. All cofactors have been omitted for the sake of clarity.

site forms a pocket surrounded by the C-terminal loop regions of α-polypeptides (Figure 3b,c). Each Sr2+ in LH1 is coordinated by the amino acid residues from two adjacent αpolypeptides. The side chain of α-Asp49 is almost certain to be a ligand (Figure 3d). This residue is also a Ca2+-coordinating ligand in the Ca-LH1-RC complex.25 The structural model refined at the current resolution suggests that the main chain oxygen of α-Asn45 in the same polypeptide and the side chain of α-Gln56 in the adjacent polypeptide also serve as ligands. The main chain oxygen of α-Tyr55 in the adjacent chain might also contribute to Sr2+ binding. There is still empty space around the metal ions that may accommodate water molecules as additional ligands. Three Sr2+-binding sites involve the amino acid residues of RC polypeptides (Figure 4a). One of them, designated Sr21, is located at the interface between the LH1 ring and the RC core, which is one of the nearest portions between the two complexes. On the basis of the structural model, this Sr2+ appears to be coordinated by two residues, L-Pro61 and LGln66, in the RC L subunit and one residue, α-Ser41, in the nearest α-polypeptide (Figure 4b). A nearby residue (α-Asp48) in a neighboring α-polypeptide may also weakly interact with this ion. The second Sr2+ (Sr22) in RC is located at the interface between the C and L subunits and surrounded by several oxygen atoms: the main chain carbonyl groups of CTyr43 and L-Thr265 and side chains of C-Asn42 and LGln172. This site is different from that of the Ca2+ bound to the RC in the Ca-LH1-RC complex, where the Ca2+ is on the opposite side of the C subunit.25 The third Sr2+ (Sr23) in RC is coordinated by the C-terminal carboxyl group of the L subunit. Strong electron densities are detected around Sr22 and Sr23, suggesting that water molecules may also be involved in the coordination. 6498


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Figure 3. Sr-binding sites in the LH1 complex. (a) Overall structure of the Sr-LH1 complex. The Sr2+ ions and BChl a macrocycles are represented by red spheres and orange sticks, respectively. (b) Top view of the expanded region marked in panel a by the dashed rectangle. (c) Side view showing the Sr- and BChl a-binding sites. (d) Close-up view of panel b showing the Sr-binding site with possible ligands.

β-BChl a and β-Trp45 of the substituted LH1s. This is also considered as a consequence of the loss of metal binding in βpolypeptides. The weakened hydrogen bonding in the substituted LH1-RC complexes is consistent with the resonance Raman result that shows a band-shift of the C3-acetyl CO stretching mode from 1637 cm−1 in the Ca-LH1-RC complex to 1643 cm−1 in the Sr-LH1-RC complex.37 There are very small changes in the relative positions of the BChl a macrocycles in LH1 as measured from the Mg−Mg distance of the neighboring BChls a. The two BChls a within an αβsubunit (protomer) tend to be slightly more separate in the Srand Ba-LH1 complexes, and this is compensated by a shortened distance for the neighboring BChls a between the αβ-subunits, leaving the overall average distance (8.78 Å) unchanged. The result may reflect a larger heterogeneity in the pigment arrangement of the substituted LH1s. The C3-acetyl CO groups of BChl a in Sr- and Ba-LH1 complexes revealed larger average values of the dihedral angle (C4−C3−C31−O32) relative to that in the Ca-LH1-RC complex. However, we have noted that there was a relatively large variation in the dihedral angles measured for both Sr- and Ba-LH1 complexes, indicating an uncertainty of this parameter in the refined models at the current resolution (3.3 Å).

LH1-RC complex from its mesophilic counterpart Alc. vinosum and are similar to those of the majority of the LH1-RC complexes in purple photosynthetic bacteria. The reduced stability makes it difficult to grow the crystals that give rise to high-resolution diffraction, but the current resolution is sufficiently high for precisely determining the positions of these metal ions in the crystal structures by anomalous scattering measurement (Figure 2). Both Sr2+ and Ba2+ are located at the same sites in the core complexes. Contrary to our anticipation, these positions in LH1 are clearly different from, though close to, the Ca sites in the Ca-LH1-RC complex with a displacement of ∼5 Å. As a result, there is a change in the metal binding environment. All the ligands identified in LH1 come from α-polypeptides. Only one residue (α-Asp49) among those that bind Ca in the Ca-LH1-RC complex is also involved in Sr2+ (or Ba2+) binding. The new metal binding pattern results in the formation of a single-ring network composed of only αpolypeptides, in contrast to the double-ring network that consists of both α- and β-polypeptides in the Ca-LH1-RC complex (Figure 5). The decreased degrees of structural integrity of the Sr(Ba)-LH1-RC complexes are considered to be responsible for the reduced thermostability and may also contribute to the change in the LH1 Qy transitions of these complexes (see below). On the other hand, the still-maintained (though weakened) LH1 network in the substituted complexes can account for another observed fact that the Sr(Ba)-LH1-RC complexes are much more stable than the metal-removed LH1RC complex.7

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DISCUSSION The Sr2+- and Ba2+-substituted LH1-RC complexes in this study exhibit LH1 Qy transitions at 888 nm (Figure 1) and decreased thermostabilities compared to that of the native Ca-LH1-RC complex from Tch. tepidum. Both features resemble those of the 6499


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Figure 4. Sr-binding sites involving the RC complex. The Sr2+ ions in LH1 have been omitted. (a) Side view of the Sr-bound LH1-RC complex showing the positions of three Sr2+ ions and their designation. (b) Close-up view of the Sr21-binding site with the ligands from two different LH1 αpolypeptides and the RC L subunit. (c) Close-up view of the Sr22-binding site with the ligands from the RC C and L subunits and a water molecule (w, magenta). (d) Close-up view of the Sr23-binding site with the ligands from the C-terminal carboxyl group of the RC L subunit and two water molecules (w, magenta).

Figure 6. Comparison of the metal-binding sites between Sr- and CaLH1 complexes. The color scheme for Sr-LH1 polypeptides is the same as in Figure 2. The BChl a macrocycles in the Sr-LH1 complex are represented as orange sticks. The polypeptides and BChl a in the Ca-LH1 complex are colored gray. The Sr2+ and Ca2+ ions are represented by red and magenta spheres, respectively. (a) Top view of superimposition of the Cα atoms in two αβ-pairs. (b) Side view from the direction perpendicular to the macrocycle plane. (c) Expanded side view from the direction parallel to the macrocycle plane.

Figure 5. (a) Side view of the overall structure of the Sr-bound LH1RC complex showing the locations of all Sr2+ ions with the presumed membrane boundary. (b) Schematic illustration showing the metalbinding network formed in Sr- and Ba-LH1 complexes. (c) Schematic illustration showing the Ca-binding network formed in LH1.

The structural origin of the difference in the LH1 Qy transition (∼330 cm−1) between the Sr(Ba)- and Ca-LH1-RC complexes is a major focus of this study. Many factors are 6500


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Table 2. Comparison of the Geometric Parameters (average values) of Hydrogen bonds, Relative Distances, and Dihedral Angles of the BChls a between the Metal-Bound LH1 Complexes from Tch. tepidum and the LH2 and LH3 Complexes from Other Species length of the H-bond to BChl O32 (Å) Sr-LH1-RC Ba-LH1-RC Ca-LH1-RC LH2 (B850)b Rps. acidophila Phaeospirillum molischianum LH3 (B820)b Rps. acidophila

Mg−Mg distance (Å)

dihedral angle of BChl C31O (deg)

distance from the metal ion to BChl C3 (Å)

α-Trp46 Nε1

β-Trp45 Nε1

longa

shorta

α-BChl

β-BChl

α-BChl

β-BChl

3.09 3.00 2.81

3.46 3.41 2.89

9.08 9.03 8.97

8.49 8.51 8.56

19.1 17.6 5.1

20.9 11.8 4.3

14.5 14.3 10.8

15.8 15.6 10.9

2.89 2.73

2.68 2.64

9.45 9.36

9.00 8.95

15.9 13.9

23.1 12.5

2.74

−

9.51

8.97

30.0

36.1

These values denote the Mg−Mg distances of neighboring BChls a within (long) and between (short) an αβ-subunit (protomer). bThese values were derived from Protein Data Bank: 1NKZ for Rps. acidophila LH2, 1LGH for P. molischianum LH2, and 1IJD for LH3. The H-bond distances were measured between BChl O32 and the closest Trp Nε1 or TyrOH. a

known to influence the excitation energy of the BChl a molecules in photosynthetic pigment−protein complexes as reviewed in the literature.36,38−42 These include both static and dynamic pigment−pigment and pigment−protein interactions. In many cases, combined effects need to be taken into account, and the structural changes that can be detected from the crystal structures are often subtle. This is also the case for this study. Here, we address several factors that are thought to be responsible for the observed LH1 Qy changes. Numerous studies have shown that the hydrogen bonding between the BChl a and LH polypeptides has a direct effect on the lowestenergy transition43−48 and can account for an ∼180 cm−1 redshift of the Qy transition.46,48 The hydrogen bonding interaction is typically a distance-dependent function.49,50 A rough estimate reveals that an increase of ∼0.5 Å in the H-bond length, as observed between the β-BChl a C3-acetyl group and β-Trp45 of the Sr(Ba)-LH1 complexes, could result in an ∼60% reduction in hydrogen bonding strength. The weakened H-bonding in the substituted LH1-RC complexes was confirmed in the resonance Raman spectra37 and may account for a maximum of half of the observed LH1 Qy change. Because the hydrogen bonding interaction can explain only part of the red-shifted Qy transition of the Ca-LH1-RC complex and the Qy transition was proposed to be relatively insensitive to the changes in geometry (e.g., position and orientation) of the pigments,40,51 other elements need to be taken into account. Slow fluctuations and static disorder in the LH complexes have been demonstrated to cause an inhomogeneous broadening of the absorption band40,52−55 and also influence its position. The best example of the former may be the observation that lowering the temperature causes a band narrowing and a red-shift of the Qy transition.36,56,57 Approximately 20 nm red-shifts were observed from room temperature to 77 K for all metal-bound and metal-removed LH1-RC complexes of Tch. tepidum.36 These were accompanied by a band narrowing of 230 cm−1 for the Ca-LH1-RC complex and 170 cm−1 for the Ba- and metal-removed LH1-RC complexes, as measured from the change in the full width at half-maximum (fwhm). The Ca-LH1-RC complex also shows the narrowest band with a fwhm of 553 cm−1 over the Sr(Ba)bound (∼640 cm−1) and metal-removed (748 cm−1) LH1-RC complexes at room temperature. These results indicate that the Ca-LH1-RC complex has a structure that is more rigid and less disordered than those of the metal-substituted and metal-

removed LH1-RC complexes and highlight an important role of the dynamic properties in electronic structure. The changes in static and dynamic disorders are considered to contribute to a large portion of the remaining LH1 Qy change. As mentioned above, the structure of the Ca-LH1-RC complex is strengthened by the double-ring LH1 network with each Ca2+ connecting both α- and β-polypeptides in an -α-Ca-β- form as shown in Figure 5c. Consequently, this results in a highly restricted molecular motion and the highest structural stability of these complexes, whereas more flexible structures are expected for the Sr(Ba)-LH1-RC complexes as the LH1 network is reduced to a single ring that is composed of the αpolypeptides only with a repeating unit of -α-Sr(Ba)-. More detailed evidence of the difference in structural integrity has been obtained (see the Supporting Information) from Stark spectroscopy,36 a titration experiment,10 differential scanning calorimetry,7 1H/2H-exchange FTIR spectroscopy,34 and isothermal titration calorimetry (manuscript to be submitted). On the basis of these results, we suggest that the Ca-LH1-RC complex may be primarily viewed as a “normal” core complex whose structure is deeply “frozen” by the Ca2+ ions. Point-charge distribution and protein polarity could induce large Qy shifts of the BChl a in a hydrophobic environment.58−61 This factor is particularly important for the systems of this study because these complexes contain a large number of divalent metal ions and the cations are located relatively close to the BChl a molecules (Figure 6). Calculations based on the known structures of RC,59 the FMO complex,62 and photosystem I63 have revealed that charged residues in the vicinity of BChl a have the most drastic effect on its Qy shift and there is a correlation between the direction of the Qy change and the location and sign of the point charge: positive charges placed near pyrrole ring I or negative charges situated near ring III result in large red-shifts, whereas reversed charges at these locations cause blue-shifts. The Ca2+ ions in the Ca-LH1 complex are located at positions with an equal distance of ∼11 Å from the rings I (C3) of α- and β-BChls a. In the substituted complexes, the Sr2+ and Ba2+ ions in LH1 moved ∼5 Å from the Ca site to an asymmetric position with distances of ∼14 and ∼16 Å to the α- and β-BChls a (Table 2), respectively. The displacement is thought to alter the electrostatic interaction network and therefore to influence the LH1 Qy transition. The impact of the long-range Coulomb interactions needs to be further examined by theoretical calculation. 6501


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Biochemistry Present Address

Finally, the torsion angle of the C3-acetyl group of BChl a has been shown by calculations to influence the Qy excitation energy.60,64 The difference in this angle was proposed to be the major cause responsible for the spectral difference between B850 in LH2 and B820 in LH3.65 However, controversies about the conclusion and the interpretation exist (see the Supporting Information).60,64 The original assignment of the Qy components in the FMO complex has been modified,41,66 and replotting the newly assigned Qy as a function of the torsion angles from the high-resolution crystal structures no longer shows a distinct correlation between the two parameters (data not shown). It was argued that the observed rotation of the acetyl carbonyl should have a very limited effect on the Qy transition, and it alone is unlikely the origin of the observed blue-shift.40 Although the structures of Sr- and Ba-LH1-RC complexes in this study show a tendency toward increased dihedral angles relative to that in the Ca-LH1-RC complex, the large variation observed at the current resolution for this value makes it difficult to draw any conclusion. It is noted that the C3-acetyl CO stretching band of the Sr-LH1-RC complex can still be observed in the resonance Raman spectrum.37 On the basis of these results, the effect of this factor needs to be reevaluated both theoretically and experimentally. In summary, the crystal structures of this work provide crucial information about the structure−function relationship of the bacterial core complex. Although the overall structures of the metal-bound LH1-RC complexes are highly conserved, pronounced differences are apparent around the metal-binding sites. The difference in the thermostability between these complexes can be attributed to the different patterns of the network formed in LH1. On the basis of the structural information at the current resolution, a combined effect of hydrogen bonding, structural integrity, and charge distribution is considered as the cause for influencing the spectral properties of the core complexes. However, at this stage, we do not know precisely the extent to which each of these factors contributes to the change in the LH1 Qy transition. The result of this study is expected to further stimulate both theoretical and experimental investigations to gain insight into the molecular mechanism for modulating the absorption properties of antenna complex.

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§

L.-J.Y.: Research Institute for Interdisciplinary Science, Okayama University, Okayama 700-8530, Japan. Author Contributions

L.-J.Y. and T.K. contributed equally to this work. Funding

This work was supported in part by Grant-in-aid for Scientific Research on Priority Areas “Structures of Biological Macromolecular Assemblies”, a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, the Takeda Science Foundation, a Grant for Basic Science Research Projects from The Sumitomo Foundation, and the Kurata Memorial Hitachi Science and Technology Foundation. L.-J.Y. gratefully acknowledges financial support in the form of a postdoctoral fellowship from Ibaraki University. T.K. is a JSPS Research Fellow. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Prof. M. T. Madigan for providing Tch. tepidum strain MC, F. Oh-hata and M. Kobayashi for their contributions to the early stage of this work, Dr. M. Unno for discussion, N. Nakamura for preparing some drawings, and Kao Corp. for kindly providing lauryldimethylamine N-oxide. X-ray diffraction data were collected at BL41XU and BL44XU of SPring-8 (Proposals 2011AB6608, 2012AB6709, 2013AB6809, 2014AB6911, 2015A1028, 2015AB6507, and 2016AB6607) and BL1A, BL17A, and NE3A of Photon Factory (Proposals 2011G514, 2013G519, and 2015G513). We are grateful to E. Yamashita, N. Matsugaki, and Y. Yamada for their support during data collection.

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ABBREVIATIONS BChl, bacteriochlorophyll; DDM, n-dodecyl β-D-maltopyranoside; DPC, n-decylphosphocholine; fwhm, full width at halfmaximum; LH1, core light-harvesting complex; LH2, peripheral light-harvesting complex; RC, reaction center; rmsd, rootmean-square deviation.

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ASSOCIATED CONTENT

(1) Madigan, M. T. (1984) A novel photosynthetic bacterium isolated from a Yellowstone hot spring. Science 225, 313−315. (2) Rowe, J. J., Fournier, R. O., and Morey, G. W. (1973) Chemical analysis of thermal waters in Yellowstone National Park, Wyoming, 1960−65. U.S. Geol. Surv. Bull. 1303, 1−31. (3) Heda, G. D., and Madigan, M. T. (1988) Thermal properties and oxygenase activity of ribulose-1,5-bisphosphate carboxylase from the thermophilic purple bacterium. FEMS Microbiol. Lett. 51, 45−50. (4) Heda, G. D., and Madigan, M. T. (1989) Purification and characterization of the thermostable ribulose-1,5-bisphosphate carboxylate/oxygenase from the thermophilic purple bacterium Chromatium tepidum. Eur. J. Biochem. 184, 313−319. (5) Moulis, J.-M., Scherrer, N., Gagnon, J., Forest, E., Petillot, Y., and Garcia, D. (1993) Primary structure of Chromatium tepidum highpotential iron-sulfur protein in relation to thermal denaturation. Arch. Biochem. Biophys. 305, 186−192. (6) Kobayashi, M., Saito, T., Takahashi, K., Wang, Z.-Y., and Nozawa, T. (2005) Electronic properties and thermal stability of soluble redox proteins from a thermophilic purple sulfur photosynthetic bacterium. Bull. Chem. Soc. Jpn. 78, 2164−2170. (7) Kimura, Y., Yu, L.-J., Hirano, Y., Suzuki, H., and Wang, Z.-Y. (2009) Calcium ions are required for the enhanced thermal stability of the light-harvesting-reaction center core complex from thermophilic

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.6b00742. Supplementary discussion, absorption spectra of the Srand Ba-substituted LH1-RC complexes at different stages of preparation, and numbers of Ca2+ ions per LH1-RC complex in the Sr2+- and Ba2+-substituted LH1-RC complexes measured by atomic absorption spectroscopy (PDF) Accession Codes

Coordinates and structure factors have been deposited in the Protein Data Bank as entries 5B5M and 5B5N.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +81-29-228-8352. 6502


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